Glaucoma is a leading cause of blindness in the United States affecting as many as 2.2 million Americans. All current glaucoma treatment strategies aim to reduce intraocular pressure (IOP). Recently our laboratory characterized a strain of mice with a targeted disruption of the Best2 gene, encoding the protein bestrophin-2 (Best2), an anion channel. In the eye, Best2 is expressed uniquely in the Non-Pigmented Epithelium (NPE) of the ciliary body. Best2-/- mice exhibit a significantly lower IOP than their wild type litermates. Surprisingly a comprehensive study of aqueous dynamics in these mice found that aqueous flow was increased and the reduced IOP observed in Best2-/- mice is due to a significantly diminished outflow resistance. Since Best2 is not expressed in outflow tissues, these effects must arise from direct communication between the inflow and outflow pathways via a pressure independent mechanism. An important goal of this application is to understand the basis of this communication. Bestrophins have a high permeability to bicarbonate, and we have recently shown that Best2 physiologically carries a bicarbonate conductance in colon goblet cells. Although carbonic anhydrase inhibitors have been used to lower IOP for many years, the role of bicarbonate in generating aqueous flow is unclear. Our preliminary data establish that a bicarbonate sensitive soluble adenylate cyclase (sAC), is expressed in NPE cells and that this sAC can regulate outflow. Yet, sAC like Best2 does not appear to be expressed in drainage tissues. Furthermore, endothelin-1 (ET1) levels are elevated in the aqueous humor of Best2-/- mice. ET1 has been shown to alter both inflow and outflow resistance potentially explaining the unusual phenotype of the Best2-/- mouse. Based on these data we propose to test the hypothesis that bicarbonate, carried by Best2 channels, is a central player in the regulation of inflow AND outflow, and that sAC mediates communication of the inflow and outflow pathways by regulating the secretion of soluble messengers such as endothelins into the aqueous humor. This will be tested in 3 specific aims. In aim 1 we determine whether bestrophin 2 serves as a bicarbonate channel in NPE cells. In aim 2, we determine the role of soluble adenylyl cyclase in the regulation of aqueous flow and intraocular pressure. In aim 3 we probe the role of bicarbonate in regulating secretion of bioactive peptides such as endothelin-1 by the ciliary body. At its conclusion this work will provide a comprehensive model of the role of bicarbonate in the physiology of the anterior chamber of the eye and could potentially lead us to new avenues for therapeutic intervention in glaucoma. PUBLIC HEALTH RELEVANCE: All current glaucoma treatments seek to reduce intraocular pressure. Deficency of the anion channel bestrophin 2 in mice results in a diminished intraocular pressure despite increased production of aqueous humor, implying the existence of a pressure independent mechanism for regulation of outflow. Understanding how bestrophin 2 functions in the cilliary epithelium and how bicarbonate regulates aqueous production and drainage will lead us to new therapies to prevent or cure vision loss due to glaucoma.